Sudan Journal of Medical Sciences Volume 17, Issue no. 3, DOI 10.18502/sjms.v17i3.12122 Production and Hosting by Knowledge E Research Article Sero-molecular Epidemiology of Hepatitis E Virus in Blood Donors, Gezira State, Sudan: A Cross-sectional Study Nassir Abakar Babiker1, Adam Dawoud Abakar2, Nawal Tagelsir Mohamed3, , Elhadi A Ahmed4, Musaab Ahmed5,6, Mohamed H Ahmed7, Abdualmoniem O. Musa8, Nadir Abuzeid9* 1Central Medical Laboratory, Wad Medani Teaching Hospital for Obstetrics & Gynecology, Wad Medani, Sudan 2Department of Medical Parasitology, Faculty of Medical Laboratory Sciences, University of Gezira, Sudan 3Department of Parasitology, NPHL Research Unit, Khartoum, Sudan 4Department of Medical Microbiology, Faculty of Medical Laboratory Sciences, University of Gezira, Sudan 5College of Medicine, Ajman University, Ajman, United Arab Emirates 6Center of Medical and Bio-allied Health Sciences Research, Ajman University, Ajman, United Arab Emirates 7Department of Medicine and HIV Metabolic Clinic, Milton Keynes University Hospital, NHS Foundation Trust, Milton Keynes, UK 8Department Microbiology, Faculty of Medical Laboratory Sciences, University of Kassala, Kassala, Sudan 9Department of Microbiology, Faculty of Medical Laboratory Sciences, Omdurman Islamic University, Omdurman, Sudan ORCID: Nadir Musa Khalil Abuzeid: https://orcid.org/0000-0003-2074-7892 Abstract Background: Hepatitis E virus (HEV) is a hepatotropic pathogen that causes significant morbidity and mortality in humans. It is an important causative agent of viral hepatitis outbreaks. This study investigates the serological and molecular prevalence of HEV in blood donors attending the Central Blood Bank in Wad Medani City in Gezira State, Sudan. Methods: The study adopted a cross-sectional descriptive design. A structured questionnaire was used to collect data concerning demographic information and risk factors associated with HEV transmission. All enrolled participants (N = 300) were screened for HEV IgG antibodies using commercial ELISA kits, then strong positive samples (N = 84) were selected and rescreened for HEV IgM and HEV RNA by RT PCR. SPSS version 24.0 was used for analysis. Results: Out of 300 male participants, 36.3% (109/300) were positive for HEV IgG. However, only one participant was IgM positive, while the HEV RNA was negative. The highest prevalence rates of the virus were 42 (44.6%) among the age group of 31–40 years, 20 (48.8%) in those who consumed food from outside, 13 (50%) in three to four multiple blood donations, and 5 (62.5%) in those who consumed water from the river source. A significant association of HEV IgG prevalence concerning the occupation of the participants being students or farmers was detected using univariate and multivariate analysis (P-value = 0.007). How to cite this article: Nassir Abakar Babiker, Adam Dawoud Abakar, Nawal Tagelsir Mohamed, , Elhadi A Ahmed, Musaab Ahmed, Mohamed H Ahmed, Abdualmoniem O. Musa, Nadir Abuzeid (2022) “Sero-molecular Epidemiology of Hepatitis E Virus in Blood Donors, Gezira State, Sudan: A Cross-sectional Study,” Sudan Journal of Medical Sciences, vol. 17, Issue no. 3, pages 376–386. DOI 10.18502/sjms.v17i3.12122 Page 376 Corresponding Author: Nadir Abuzeid; email: nadirabuzeid@oiu.edu.sd Received 22 August 2021 Accepted 5 August 2022 Published 30 September 2022 Production and Hosting by Knowledge E Nassir Abakar Babiker et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Editor-in-Chief: Prof. Nazik Elmalaika Obaid Seid Ahmed Husain, MD, M.Sc, MHPE, PhD http://www.knowledgee.com https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ Sudan Journal of Medical Sciences Nassir Abakar Babiker et al Conclusion: High prevalence of HEV IgG was demonstrated among the healthy blood donors in this study. Given the possibility of HEV transmission by transfusion from donors to recipients, we recommend that routine screening for HEV should be adopted by blood banks in Sudan. Keywords: HEV IgG, blood donors, HEV risk factors, HEV IgM, HEV RT PCR, Sudan 1. Introduction Hepatitis E virus (HEV) initially belonged to the family Caliciviridae, but recently after the introduction of molecular techniques, the virus has been reclassified and placed in the Hepeviridae family [1]. Although the virus is serotypically homogenous, it has been subdivided into four genotypes: genotypes 1 and 2 are mainly found in humans, whereas genotypes 3 and 4 represent porcine HEVs but can also infect humans [2]. In addition, the family of Hepeviridae is phylogenetically divided into two genera, with four species in genus Orthohepevirus [3]. HEV prevalence rate in Africa is variable. It was reported to be 45.3% in Egypt [4], 5.4% and 22% in Tunisia [5, 6], and 19.1% in Burkina Faso blood donors [7]. Studies conducted among risk groups revealed the variable distribution of serological markers of HEV [8–10]. Transmission through pooled platelet concentrates was concluded in some studies in Germany [11]. In addition, HEV RNA-positive blood donations from healthy blood donors were observed in Japan [12]. Studies from Japan and Europe showed that a few cases of HEV were transmitted by blood transfusion, and retrospective follow-up of the transfused patients revealed elevated transaminase levels and signs of fulminant hepatitis [13–15]. A retrospective study of transfusion recipients suggested that in HEV-1-endemic areas, HEV could be transmitted by blood transfusion [16]. A similar conclusion was demonstrated in another study, in which some recipients exhibited HEV reactivity [17] after blood transfusion. In the Egyptian population, a study revealed that 80% were HEV IgG positive. However, only 0.26% were found to be HEV RNA positive [18]. A study aiming at molecular detection of HEV RNA revealed negative results among 239 Ghanaian blood donors [19]. Similar results were obtained from American plasma donations [20]. Low rates of HEV RT-RNA were detected in studies conducted among blood donors in Sweden (0.012%), Germany (0.022%) [20], and Egypt [18]. The HEV RNA seems quite difficult to detect and this may require a very large sample size with highly sensitive RT-PCR methods. In a study of blood donations in China, 30 out of 44,816 DOI 10.18502/sjms.v17i3.12122 Page 377 Sudan Journal of Medical Sciences Nassir Abakar Babiker et al donations (0.07%) were HEV RNA-positive [21]. In addition, low HEV RNA production rates were concluded by Fischer et al., where they could only detect 7 (0.58%) HEV RNA among 1203 (13.55%) positive HEV IgG [22]. Furthermore, Nassir et al. revealed a negative result concerning the HEV-RNA among hemodialysis patients with a positive HEV seroprevalence [23]. On the other hand, the viral distribution concerning the gender and age groups revealed some variation among the studies. For example, a study conducted in Iran showed a difference in HEV IgG by age but not gender [24]. A higher HEV IgG seroprevalence rate of 26.7% concluded by Ahmed et al. among Sudanese blood donors that are significantly associated with a young age group might raise the potential risk for HEV transmission in recurrent blood transfusion by those donors. 2. Materials and Methods 2.1. Study design and samples collection The study was a cross-sectional, hospital-based descriptive study conducted among blood donors attending the central blood bank in Wad Medani city, the capital of Gezira state, Sudan. This blood bank provides blood donation services to all localities of Gezira State. Therefore, donors in this study could be representatives of the population in Gezira State. After testing negative during the routine screening of HBV, HCV, syphilis, and HIV for the blood transfusion, 300 blood donors were recruited for this study. Before the sample collection, informed consent was taken from all participants and the demographic data, clinical data, and risk factors associated with HEV transmission were documented using a structured questionnaire. About 5 ml blood was collected from each participant in an EDTA container using standard techniques. The plasma was separated into two aliquots, then stored at –40ºC until used for HEV serological screening by ELISA and HEV RNA by RT-PCR. 2.2. Serological screening for HEV The screening was performed using ELISA for HEV IgG and HEV IgM. For the inter- pretation of the negative results for HEV IgG or HEV IgM, the positive results, and the equivocal results, the cut-off value was set as <1, >1, and 0.9–1.1, respectively, DOI 10.18502/sjms.v17i3.12122 Page 378 Sudan Journal of Medical Sciences Nassir Abakar Babiker et al according to the manufacturer’s instructions. The equivocal reading was later retested for confirmation. 2.3. Molecular detection for HEV-RNA The Primer design𝑇𝑀 genesis® (UK manufacture) kits were used for the extraction and real-time PCR of HEV-RNA detection. The kits are specifically designed for in vitro highly purified RNA extraction and quantification of HEV genomes. The primers and probe sequences in the kit had 100% homology with a broad range of HEV sequences based on comprehensive bioinformatics analysis. Both RNA extraction and real-time PCR were carried out at the National Public Health Laboratory, Khartoum, Sudan, using an RT-PCR Thermo cycler (Rotor gene 6000, Germany). The DNA/RNA Extraction Kit solution (EXP date: 17/10/ 2017, Batch No, 66606, G25493, 66601, 66605, P15079S, 66628, 66622, SHB68127V, and 66596) and the HEV-RNA-specific primer and probe mix (Serial number JN 160305-49345, Batch number PD2879, Expiry date 17.10.2017) were used according to the manufacturer’s protocol. 2.4. Statistical analysis Data were analyzed using the Statistical Package for Social Science (SPSS), version 24.0. Descriptive statistics for HEV prevalence and different variables are illustrated in Tables 1 and 2. Table 1 demonstrates the frequencies among the localities and Table 2 presents the frequencies regarding the demographic and risk factors for virus transmission. The Chi-square test for categorical variables was calculated. Further, for the logistic regression of univariate and multivariate analysis, an odds ratio (OR) with a 95% confidence interval (CI 95%) was calculated, and the statistical significance was defined as P < 0.05. 3. Results 3.1. Epidemiological and demographical analysis of the study groups All blood donors recruited for the current study were male, aged between 18 and 50 years and belonging from different localities of Gezira State, Sudan (Tables 1 & 2). Out of the 300 blood donors, 109 (36.3%) were HEV IgG-positive and 191 (63.7%) were negative (Figure 1). The highest HEV seroprevalence was found among participants in the age DOI 10.18502/sjms.v17i3.12122 Page 379 Sudan Journal of Medical Sciences Nassir Abakar Babiker et al group of 31–40 years, with a percentage of 44.6%, and the lowest rate was predicted among the age group of 18–30 years with a prevalence of 32.5% (Table 2). 3.2. HEV IgG seroprevalence and the associated risk factors local- ities The distribution of seroprevalence of HEV IgG among the localities were as follows: 34 (31.5%) in Wad Medani locality, 30 (34.1%) in South Gezira locality, 16 (59.3%) in Managil, 15 (37.5%) in East Gezira, 10 (41.7%) in Um Algura, and 4 (30.7%) in Hasahesa localities (P > 0.5). 3.2.1. Occupations Higher prevalence of the HEV IgG was calculated among eight (47.1%) farmers, while no prevalence of the virus was found among health workers and policemen (Table 2). A significant association was calculated among students and farmers regarding the HEV IgG distribution and the factors of the disease in univariate and multivariate analysis (P = 0.007), Table 2. 3.2.2. Animal contact The HEV IgG seroprevalence regarding the risk of contact with the animals out of 300 blood donors resulted in 26 (46.3%) for blood donors with animal contact and 83 (34%) with no animal contact (P= 0.083), Table 2. 3.2.3. Food source The HEV IgG prevalence concerned the sites of food sources out of the 300 blood donors resulted in 74 (32.6%) at home, 20 (48.8%) outside the home, and 15 (46.9%) at home and outside the home, Table 2. 3.2.4. Water source Out of the 300 blood donors, 100 (36.2%) HEV IgG-positive used wells as water sources, 5 (62.5%) used rivers, and only 4 (25%) used Hafeer (a hole in the ground used to collect water during the rainy season for using it later, Table 2. DOI 10.18502/sjms.v17i3.12122 Page 380 Sudan Journal of Medical Sciences Nassir Abakar Babiker et al 3.2.5. The number for blood donation The HEV IgG prevalence concerning the numbers for the respondents who donated blood resulted in 89 (35.6%) donating blood once or twice, 13 (50%) donating three to four times, and 7 (29.2%) donating more than four times, Table 2. 3.2.6. HEV IgM and HEV RNA analysis Out of the 84 blood donors with HEV IgG strong positive samples, only 1.2% (1/84) were positive for HEV IgM, while no HEV RNA was predicted amongst any of them by using an RT-PCR assay. 0 50 100 150 200 250 A m o u n t o f D o n o rs HEV IgG(+) HEV IgG(-) 63.7% 36.3% Figure 1: The distribution of HEV IgG among blood donors (N = 300). Table 1: The HEV IgG distribution among blood donors according to Gezira State localities (N = 300). Variables HEV IgG Univariate Multivariate Negative Positive (%)N = 109 Total OR CI 95% P-value OR CI 95% P-value Wad Medani 74 34 (31.5) 108 0.967 0.278–3.362 0.958 0.854 0.240–3.030 0.806 Um Alqura 14 10 (41.7) 24 0.622 0.149–2.601 0.516 0.587 0.139–2.475 0.468 South Algezira 58 30 (34.1) 88 0.859 0.244–3.022 0.813 0.805 0.227–2.857 0.737 East Algezira 25 15 (37.5) 40 0.741 0.194–2.830 0.661 0.664 0.171–2.58 0.555 Almanagil 11 16 (59.3) 27 0.306 0.075–1.246 0.098 0.305 0.074–1.252 0.099 AlHasahisa 9 4 (30.7) 13 0.306 0.075–1.246 0.098 0.305 0.074–1.252 0.099 Total 191 109 (36.3) 300 OR, odds ratio; C, 95% confidence interval; HEV, hepatitis E virus. DOI 10.18502/sjms.v17i3.12122 Page 381 Sudan Journal of Medical Sciences Nassir Abakar Babiker et al Table 2: The HEV IgG distribution among blood donors according to demographic and risk factors. Variables HEV IgG Univariate Multivariate Negative Positive (%)N = 109 Total OR CI 95% P-value OR CI 95% P-value Age group (yr) 18-30 135 65 (32.5%) 200 1.038 0.185–5.817 0.966 0.876 0.152–5.028 0.882 31-40 52 42 (44.6%) 94 0.619 0.108–3.546 0.59 0.544 0.093–3.178 0.499 >40 4 2 (33.3%) 6 0.619 0.108–3.546 0.59 0.544 0.093–3.178 0.499 Occupation Casual Workers 117 87 (42.6%) 204 1.195 0.443–3.223 0.724 1.086 0.397–2.97 0.873 Drivers 16 6 (27.27%) 22 0.579 0.369–5.946 0.579 1.345 0.331–5.471 0.678 Policemen 8 0 8 2164 0.000–0.000 0.997 2033 0.000–0.000 0.997 Engineers 7 1 (12.5%) 8 6.222 0.623–62.159 0.12 6.222 0.618–62.6 0.121 Teachers 3 2 (40%) 5 1.333 0.176–10.120 0.781 1.112 0.144–8.60 0.919 Health workers 2 0 2 21643 2164–216437 – 1805 1805–1805 – OR, odds ratio; C, 95% confidence interval; HEV, hepatitis E virus. 4. Discussion Studies assessing the risk of HEV transmission through blood transfusion are relatively fewer than others conducted for HCV and HBV. Researchers from Japan and Europe indicated that a few cases of HEV were transmitted by blood transfusion and later developed elevation of transaminase levels and fulminant hepatitis [13–15]. Another study showed that some recipients exhibited HEV reactivity after a blood transfusion from an HEV-infected donor [17]. According to our study, HEV IgG seroprevalence among healthy blood donors was 36.3%. This result was higher than that revealed by Ahmed et al. [25] among Sudanese blood donors (26.7%) and lower than that reported among Egyptian blood donors (45.3%) [4], but higher than what was reported by other studies from other African countries [6, 7, 19]. Based on the results of this study, the highest prevalence of HEV IgG seroprevalence was among those aged 31–40 years, 42 (44.6%). This finding could reflect a higher probability of virus exposure among this age group, which might be due to occupational hazards (e.g., farmers and animal contact as approved in the study) or other factors, for instance, this age group might be more likely to consume food outside the home which is a known risk factor for foodborne infections. However, in other studies, such as that conducted by Adjei et al., HEV seroprevalence was more among the elderly than in younger persons. It should be noted that statis- tically there was no significant association between HEV prevalence and age group among blood donors in our study and this is consistent with a study conducted in the Netherlands [9]. DOI 10.18502/sjms.v17i3.12122 Page 382 Sudan Journal of Medical Sciences Nassir Abakar Babiker et al It was obvious, in our study, that those people who drank the river water were at a higher risk of getting infected (5; 62.5%), compared to those drinking from well sources (100; 36.2%) and Hafeer (4; 25%). Some evidence of significant association regarding the virus prevalence concerning the water sources revealed the highest risk of river sources compared to the well source and the lowest risk of Hafeer sources, OR = 5.000, P = 0.084 and OR = 5.145, P = 0.081; P = 0.081, OR = 5.000, CI 95% = 0.806–31.002, respectively, Table 2. This might be due to a higher likelihood of contamination of the rivers by animal and human products than the wells. Furthermore, our study identified those with a previous history of multiple blood donations have a higher HEV IgG seroprevalence 13 (50%). This is consistent with the results from other studies [13–15, 17] and as the majority of them are young, this leads to the possibility of later donations in the future which could be an additional risk for transmission. Although, the HEV IgM seropositive in this study was the only donor; therefore, it was a limitation for our study to carry out statistical HEV IgM interpretation. However, the result still increases the probability to transfuse HEV-contaminated blood to those patients with compromised immunity [16]. HEV RNA concluded in a negative result for all blood donors, and that might be for our sample size limitation to carry out the HEV RT-PCR, some authors obtained a similar result for a negative HEV-RNA [9, 20, 24]. This study is novel in Gezira State, Sudan that showed relations and association of risk factors for HEV and blood transfusion. The limitation of the study was the small sample size and the eclipsing period of HEV, and the collection time of the viruses and ages; therefore, we could not reliably assess the true prevalence of IgM- and RNA-positive cases. 5. Conclusion The results of this study could suggest a high probability of HEV transmission through blood transfusion as reflected by the high HEV seroprevalence among healthy donors. The risk factors that could be identified in this study include the occupation (mainly farmers), animal contact, and river sources for drinking. Acknowledgments The authors are grateful to all those who participated in this study, namely, the staff of central blood bank in Wad Medani City, the capital of Gezira state, Sudan, the DOI 10.18502/sjms.v17i3.12122 Page 383 Sudan Journal of Medical Sciences Nassir Abakar Babiker et al National Public Health Laboratory, Khartoum, Sudan, and the Department of Medical Microbiology, Faculty of Medical laboratory of Sciences, Gezira University. Ethical Considerations Not applicable. Competing Interests None declared. Availability of Data and Material The dataset generated during this study is available from the corresponding author on reasonable request. Funding None. References [1] Emerson, S., Anderson, D., Arankalle, A., Meng, X., Purdy, M., Schlauder, G., & Tsarev, S. (2004). Hepevirus. Virus Taxonomy, 853–857. [2] Modrow, S., Falke, D., Truyen, U., & Schätzl, H. (2013). Viruses with single-stranded, segmented, negative-sense RNA genomes. Molecular Virology. Springer. [3] Smith, D. 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Hepatitis E virus seroprevalence among blood donors in Bushehr, South of Iran. Hepa Month, 15(11). [25] Ahmed, A. B., & Hamedelnil, Y. F. (2015). Seroprevalence of hepatitis E virus among blood donors in Omdurman locality, Sudan. American Journal of Research Communication, 3(5), 252–258. DOI 10.18502/sjms.v17i3.12122 Page 386 Introduction Materials and Methods Study design and samples collection Serological screening for HEV Molecular detection for HEV-RNA Statistical analysis Results Epidemiological and demographical analysis ofthe study groups HEV IgG seroprevalence and the associated risk factors localities Occupations Animal contact Food source Water source The number for blood donation HEV IgM and HEV RNA analysis Discussion Conclusion Acknowledgments Ethical Considerations Competing Interests Availability of Data and Material Funding References